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Abstract:

A novel dosing regimen for the administration of botulinum toxin based on
the pattern, quantity, and location of neuromuscular junctions in the
target tissue. Because the number of neuromuscular junctions in a target
tissue remains generally stable throughout life and because the
pharmacological effect of botulinum toxin is localized at the
neuromuscular junction, dosing efficacy is unaffected by muscle mass, age
of the patient, or body weight.

Claims:

1-20. (canceled)

21. A method for dosing botulinum toxin in a target muscle of humans who
are at the extremes of weight distribution curves with high and low body
mass comprising the steps of: a) determining the distribution pattern and
location of neuromuscular junctions (NMJ) in said target muscle to
ascertain the amount of botulinum toxin to be used on said target muscle
through injection sites; b) determining the mass of a target muscle; c)
diluting a solution containing the botulinum toxin so that dilution of
the solution by volume increases in an amount proportionate to the mass
of said target muscle as a ratio of its mass in relation to the mass of
another base reference muscle with increasing body weight; d) injecting
doses of said diluted botulinum toxin solution adjacent locations of said
neuromuscular junctions of said target muscle.

23. A method for dosing botulinum toxin A in a human target muscle so
that toxin distribution to non-targeted tissue is limited comprising the
steps of: a) determining the mass of a target muscle relative to the mass
of a lateral gastrocnemius muscle; b) diluting a solution containing
botulinum toxin A so that dilution of the toxin increases in an amount
proportionate with body weight and relative to the ratio of the mass of
said lateral gastrocnemius muscle and the mass of said target muscle; c)
determining the distribution pattern and location of neuromuscular
junctions (NMJ) in said target muscle; and, d) injecting doses of
botulinum toxin A diluted solution adjacent said neuromuscular junctions
relative to the location and quantity of said neuromuscular junctions in
said target muscle to obtain maximum treatment.

24. The method of claim 21 wherein said botulinum toxin is botulinum
toxin is taken from a family of botulinum toxins designated A, B, C, D,
E, F and G.

45. The method of claim 21 wherein the location of said neuromuscular
junctions of said target muscle is determined using ultrasound
localization.

26. The method of claim 21 wherein said doses are delivered within a
range of about 0.1 cm to about 3.0 cm to the area of the target muscle
containing said neuromuscular junctions.

27. The method of claim 21 wherein said target muscle is a unipennate
muscle taken from the group consisting of the opponens pollicis,
semitendinosus, and brachioradialis, and said neuromuscular junctions are
distributed about a transverse midline of said target muscle.

28. The method of claim 21 wherein said target muscle is a unipennate
gracilis muscle and said neuromuscular junctions are distributed about
two transverse lines on said target muscle.

29. The method of claim 21 wherein said target muscle is a bipennate
converging muscle taken from the group consisting of the gastrocnemius
and biceps brachii, and said neuromuscular junctions are distributed in a
substantially inverted U-shaped pattern on said target muscle.

30. The method of claim 21 wherein said target muscle is a soleus muscle
and said neuromuscular junctions are distributed along the length of the
muscle fibers.

31. The method of claim 21 wherein said target muscle is a rectus femoris
and said neuromuscular junctions are distributed about two longitudinal
lines running along the length of said target muscle.

32. The method of claim 21 wherein said target muscle is a deltoid muscle
and said neuromuscular junctions are distributed irregularly on said
target muscle.

33. The method of claim 23 wherein said target muscle is a unipennate
muscle taken from the group consisting of the opponens pollicis,
semitendinosus, and brachioradialis, and said neuromuscular junctions are
distributed about a transverse midline of said target muscle.

34. The method of claim 23 wherein said target muscle is a unipennate
gracilis muscle and said neuromuscular junctions are distributed about
two transverse lines on said target muscle.

35. The method of claim 23 wherein said target muscle is a bipennate
converging muscle taken from the group consisting of the gastrocnemius
and biceps brachii, and said neuromuscular junctions are distributed in a
substantially inverted U-shaped pattern on said target muscle.

36. The method of claim 23 wherein said target muscle is a soleus muscle
and said neuromuscular junctions are distributed along the length of the
muscle fibers.

37. The method of claim 23 wherein said target muscle is a rectus femoris
and said neuromuscular junctions are distributed about two longitudinal
lines running along the length of said target muscle.

38. The method of claim 23 wherein said injected doses of botulinum toxin
A are injected within 3.0 cm of said mircomuscular functions.

39. The method of claim 21 wherein said base reference muscle is a
lateral gastrocnemius.

40. A method for dosing botulinum toxin in a target muscle of humans
comprising the steps of: a) selecting a botulinum toxin taken from a
family of botulinum toxins designated A, B, C, D, E, F and G having
maximum effect on a target muscle selected for treatment; b) determining
the distribution pattern and location of neuromuscular junctions (NMJ) in
said target muscle to ascertain the amount of botulinum toxin to be used
on said target muscle through injection sites; c) determining the mass of
a target muscle; d) diluting a solution containing the botulinum toxin so
that dilution of the solution by volume increases in an amount
proportionate to the mass of said target muscle as a ratio of its mass in
relation to the mass of another base reference muscle with increasing
body weight; and e) injecting doses of said diluted botulinum toxin
diluted solution at locations within 3.0 cm of said neuromuscular
junctions of said target muscle in a volume to obtain maximum treatment.

REFERENCE TO SEQUENCE LISTING, A TABLE OR A COMPUTER PROGRAM LISTING
COMPACT DISC APPENDIX

[0003] None.

BACKGROUND OF THE INVENTION

[0004] 1. Field of Invention

[0005] This invention relates to a dosing protocol for the administration
of botulinum toxin that maximizes efficacy and specificity while
minimizing the likelihood of overdosing and undesirable side effects of
botulinum toxin treatment.

[0006] 2. Background of the Invention

[0007] Botulinum toxins, in particular botulinum toxin type A, have been
used in the treatment of a number of neuromuscular disorders and
conditions involving muscular spasm as well as in cosmetic procedures;
for example, strabismus, blepharospasm, spasmodic torticollis (cervical
dystonia), oromandibular dystonia and spasmodic dysphonia (laryngeal
dystonia). The toxin binds rapidly and strongly to presynaptic
cholinergic nerve terminals and inhibits the exocytosis of acetylcholine
by decreasing the frequency of acetylcholine release thereby reducing or
eliminating the activation of postsynaptic muscles, nerves, or effector
tissues. This results in local paralysis and hence relaxation of the
muscle afflicted by spasm.

[0008] The term botulinum toxin as used herein is a generic term embracing
the family of toxins produced by the anaerobic bacterium Clostridium
botulinum and, to date, seven immunologically distinct toxins have been
identified. These have been given the designations A, B, C, D, E, F and
G. For further information concerning the properties of the various
botulinum toxins, reference is made to the article by Jankovic & Brin,
The New England Journal of Medicine, pp 1186-1194, No 17, 1991 and to the
review by Charles L Hatheway, Chapter 1 of the book entitled Botulinum
Neurotoxin and Tetanus Toxin Ed. L. L. Simpson, published by Academic
Press Inc. of San Diego Calif. 1989, the disclosures in which are
incorporated herein by reference.

[0009] The neurotoxic component of botulinum toxin has a molecular weight
of about 150 kilodaltons and is believed to comprise a short polypeptide
chain of about 50 kD which is considered to be responsible for the toxic
properties of the toxin, and a larger polypeptide chain of about 100 kD
which is believed to be necessary to enable the toxin to penetrate the
nerve. The "short" and "long" chains are linked together by means of
disulphide bridges.

[0010] Intramuscular injections of botulinum toxin A are generally used to
balance muscle forces across joints, to diminish or decrease painful
spasticity, to decrease deforming forces through selective motor
paralysis, to diminish neuropathic and nociceptive pain, to diminish
dystonic contractures, to decrease muscle deformation after injury or
surgery, and to diminish sweating. The target organelles contain soluble
NSF attachment receptor (SNARE) proteins and neurotransmitter-containing
vesicles which require these SNARE proteins for fusion of the vesicle to
the cell membrane and release of neurotransmitter. Targets include
neuromuscular junctions, sweat glands, vascular beds and nociceptors.

[0011] Therapeutic use of these toxins represents a somewhat unique
pharmacokinetic profile. In order for toxin to produce its desired
action, it must not only be delivered to the target tissue, e.g. muscle
(usually by direct injection), but it must also bind to terminal portions
of nerves innervating the target tissue (i.e. the neuromuscular
junction), and be transported across the presynaptic terminal membrane
into the intracellular domain where the active molecule is cleaved from
the binding portion of the divalent complex. Then the active molecule
must bind irreversibly and enzymatically inactivate molecules in the
nerve terminal specific for neurochemical transmission. Thus the toxin
molecules are not delivered systemically to distribute throughout the
body. The ultimate target is not a specific muscle or organ but rather
molecules located in specific nerves which innervate the target tissue
within an anatomically defined region of the target tissue or muscle. For
example, within skeletal muscle fibers, nerves do not uniformly
distribute through the muscle but rather the terminals of the nerves are
restricted to a certain region of the muscle. In the case of muscle
fibers, prior research has shown that different muscles have different
numbers of neuromuscular junctions and the total number of these
neuromuscular junctions is not dependent on the mass or volume of the
muscle or the individual but rather on other factors such as the function
of the muscle fibers.

[0012] Current recommendations and dosing regimens are empirical and
utilize dosage based upon bodyweight in, for example, the management of
cerebral palsy and in orthopaedic uses. With specific regard to its use
in children, the use of botulinum toxin in the management of cerebral
palsy and in orthopaedic usage is based on the size and weight of the
growing child, rather than age, to insure safety since overall toxicity
data was based upon units per kilogram of body weight in primates. U.S.
Pat. No. 6,395,277 issued 28 May 2002 shows a dosing regimen for the
treatment of cerebral palsy, noting that dosing should occur "preferably
. . . in the region of the neuromuscular junction" according to "the
number of muscle groups requiring treatment, the age and size of the
patient." Similar dosing regimens base relative dosages upon the size of
muscle.

[0013] Historically, dosage recommendations for administration of
botulinum toxin has been an imprecise science. Recommendations have been
made on the basis of body weight, body surface area, size or volume
occupied by a specific muscle, etc. The overreaching goal for each of
these therapeutic or cosmetic uses of botulinum toxins is that the toxin
be administered in a dosage and volume appropriate to achieving the
desired response while remaining localized within the desired specific
region of injection. Because the ultimate site of toxin action is nerve
junctions within certain regions of the target tissue, over- and
under-dosing remains a significant challenge. Administration of too high
an absolute dose (total number of toxin molecules relative to the total
number of neuromuscular junction targets) or too high a volume of
injection might produce adverse reactions related to diffusion of the
toxin. Diffusion of the toxin into undesired areas could produce
inappropriate paralysis or pathophysiological responses. Too high a dose
will produce the desired effect of tissue paralysis but also result in
toxin distribution to non targeted tissues thereby causing an unintended
loss of physiological function in these regions. Additionally, delivery
of supraoptimal toxin doses presents an undesired immunological challenge
which may cause reduced effectiveness on subsequent administrations of
the toxin. When a large volume of toxin is delivered, it is likely that
toxin molecules will diffuse to distant targets resulting in the dilution
of the effect of the toxin at the desired target and inappropriately
exposing other regions to the toxin. In a large volume dosing scenario, a
higher overall dose of toxin would be required at a later time to
overcome the dilution effect thus increasing the exposure of other
tissues. In these cases where inappropriate doses or volumes are used,
not only may patient safety be compromised but the cost of the procedure
is increased due to wasted toxin or treatment of unanticipated
pharmacological outcomes.

[0014] Perhaps the most obvious examples of this inappropriate dosage are
delivery of toxin based on body weight to individuals who are at the
extremes of weight distribution curves. The toxin acts at the
neuromuscular junction and the quantity of the aforementioned junctions
does not change proportionately with changes in body mass. Hence, in
these cases, individuals with high and low body mass would receive
inappropriately high or low doses, respectively.

[0015] Various recommendations have demonstrated clinical usefulness but
fail to address that 1) the toxin acts at the neuromuscular junction, and
2) the number of neuromuscular junctions varies from muscle to muscle,
and 3) the number of neuromuscular junctions tends not to vary as a
person ages. Neuromuscular junctions for individual muscles are not
directly proportional to muscle mass or volume. Rather, the distribution
of neuromuscular junctions varies from muscle to muscle and the number of
neuromuscular junctions is affected minimally by age and total body
weight. The existing dosage recommendations are clinically efficacious in
50 to 70 percent of patients, namely large toddlers and adolescents, but
may underdose infants and small toddlers and overdose heavy children,
teenagers, and adults. What is needed are more precise dosing methods to
delineate optimal number of units, volume, and injection sites for
individual muscles, thereby improving efficacy, minimizing protein
antigen load and subsequent antibody formation, and decreasing costs.

SUMMARY OF THE INVENTION

[0016] The present invention is a novel dosing method for botulinum toxin
based on the number and distribution of neuromuscular junctions in the
target muscle. It includes determining the mass of the target muscle,
determining the distribution and location of neuromuscular junctions in
that muscle, and injecting an appropriate therapeutic dose of botulinum
toxin in the vicinity of and according to the quantity of neuromuscular
junctions in the muscle. A dosing regimen based on the quantity of
neuromuscular junctions in the aforementioned tissue ensures efficacy,
while minimizing possible side effects and minimizing cost by ensuring
that only that amount of toxin necessary to achieve the desired effect is
used.

[0017] It is an object of this invention to provide a safe dosing method
for botulinum toxins;

[0018] It is another object of this invention to provide an efficacious
method for dosing botulinum toxins;

[0019] It is still another object of this invention to provide a minimally
invasive means of dosing botulinum toxins;

[0020] It is yet another object of this invention to provide a cost
effective dosing method for botulinum toxins; and,

[0021] It is an object of this invention to provide a simple, easily
complied with dosing method for the use of botulinum toxins.

[0028]FIG. 7 shows a rectus femoris muscle with two bands of
neuromuscular junctions running along its length; and,

[0029]FIG. 8 shows a deltoid muscle with an irregular pattern of
neuromuscular junctions.

[0030] These and other objects, advantages, and novel features of the
present invention will become apparent when considered with the teachings
contained in the detailed disclosure along with the accompanying
drawings.

DESCRIPTION OF THE INVENTION

[0031] While the invention is described in connection with certain
preferred embodiments, it is not intended that the present invention be
so limited. On the contrary, it is intended to cover all alternatives,
modifications, and equivalent arrangements as may be included within the
spirit and scope of the invention as defined by the appended claims.

[0032] The invention is a novel dosing method for botulinum toxin based on
the quantity and distribution of neuromuscular junctions in a target
tissue. Previous recommendations for dosing were based on, for example,
body mass and/or age. In the present invention, therapeutic dosing is
based on 1) the quantity and distribution of neuromuscular junctions and
2) the volume of liquid or other carrier material in which that dose is
delivered to the target tissue. This results in decreased incidences of
under or over-dosing, minimized direct costs of administrating the
substance due to the more efficient use of the toxin itself, and
minimized indirect costs resulting from the medical costs avoided by
eliminating the likelihood of anaphylaxis and immuno-challenge resulting
from too high a dose. Ideal dose in units is therefore based upon the
number of NSF attachment receptor (SNARE) containing organelles (i.e.,
neuromuscular junctions to be blocked); the volume or concentration
calculated from the muscle mass; and the number of injection sites is
dictated by the length and width of the target tissue.

[0033] As shown in the Dose Response Recovery Graph of FIG. 2, because its
efficacy is dependant on the quantity of neuromuscular junctions in the
target tissue, dosing of botulinum toxin exhibits clear maximum dosing
behavior beyond which an increased dose shows no appreciable change in
effect. The neuromuscular junctions of the target tissue are saturated
such that additional availability of toxin produces no additional effect.
The graph of FIG. 3, however, shows clearly that proper titration of the
dose is important. While suboptimal amounts of toxin obviously produce
lower degrees of relaxation, supraoptimal doses produce similarly reduced
results. It is believed that the reduced results occur because toxin
molecules diffuse away from the target site resulting in the dilution of
the effect of the toxin at the desired target and inappropriately expose
other regions to the toxin. Hence determination of the most efficacious
dosage for a target muscle is critical.

[0034] Botulinum toxin A is produced by Allergan Pharmaceuticals as
BOTOX® and by Ipsen Limited Phannaceuticals as Dysport®. Each
vial of BOTOX® contains 100 units of C. botulinum type A neurotoxin
complex, 0.5 mg of human albumin, and 0.9 mg of sodium chloride as a
vacuum-dried frozen powder that requires reconstitution. One unit of
BOTOX® is equal to the median intraperitoneal lethal dose (LD50) in
Swiss-Webster mice weighing 18 to 20 g. The LD50 for Botox® has been
calculated in primates at 39 to 56 units/kg body wt. However, the exact
lethal dose in humans is unknown. The calculated human LD50 of 59 units
is based on an extrapolation of data. Dysport® clostridium botulinum
type A toxin-hemogglutinin complex is available in 500-unit vials.
Dysport® units of activity equal 1 mouse LD50 based on their specific
assay technique and is sometimes referred to in nanograms, with 1
nanogram equal to 40 units. In the United Kingdom and many other
countries, it is approved and labeled for multiple indications, including
spasticity of the arm in patients following stroke, dynamic equinus foot
deformity due to spasticity in ambulant pediatric cerebral palsy
patients, two years of age or older, spasmodic torticollis,
blephorospasm, and hemifacial spasm. With regard to cerebral palsy,
Dysport® dosing is recommended as "30 units/kg body weight divided
between both calf muscles.".

[0035] Existing clinical data supports that BOTOX® and Dysport®
potencies are different; one BOTOX® unit is equal to 2 to 4
Dysport® units. Units are not interchangeable between companies or
toxin types using package guidelines and suggested dilution tables.

[0036] Both BOTOX® and Dysport® are reconstituted in injectable
physiologic saline prior to intramuscular injection. Both the volume of
fluid and number of units of drug must be considered when preparing the
toxin for injection. Dosage is defined in absolute terms, based on the
number of units per target muscle diluted to volume based on the size of
the structure to be injected and quantity and distribution of
neuromuscular junctions. The number of units to be injected is calculated
by the quantity of neuromuscular junctions to be neutralized, and the
volume is determined by the mass of the target muscle, and the number of
injection sites by the anatomic distribution of the neuromuscular
junctions. Once the appropriate number of active toxin molecules (units)
for a given muscle is determined, the dose in units remains constant and
the volume and number of injection sites is adjusted based upon growth
and anatomy. For example, there are an estimated pikamole of active toxin
molecules in 100 units of BOTOX® and an estimated 250,000
neuromuscular junctions in the human biceps brachii. Hence, there are
sufficient active toxin molecules to block effectively all neuromuscular
junctions of the "target" muscle. The toxin is thereafter injected within
the muscle or skin as close to the neuromuscular junctions (or other
SNARE-containing organelle) as possible using ultrasonography to localize
their position.

[0037] Visualization of extremity and trunk muscles is performed reliably
using linear probe ultrasonography with a frequency of 5-12 Mhz. For
injection localization, linear beam applications better define and
delineate the anatomic relationships between muscles, tendons or bones.
Higher frequencies are recommended for the localization of the
superficial muscles or layers, while lower frequencies may be used for
deep structures. The muscles are covered by the epimysium which is the
connective tissue that surrounds the entire muscle. The epimysium extends
into the muscle to become the perimysium, which divides the fascicle into
muscle fibers. The perimysium and the muscular fascicles can be
identified because the muscular bundles are hypoechoic (less bright)
while the epimysium and perimysium appear as hyperechoic structures. On
longitudinal scanning, the fascia is depicted as a fibrillar hyperechoic
sheath surrounding the muscle.

[0038] There are approximately 250,000 neuromuscular junction in the human
biceps brachii muscle. Other human extremity muscles (e.g., the lateral
and medial head of the gastrocnemius) have a similar neuromuscular
junction density. The total dosage of botulinum toxin (i.e., the absolute
number of toxin molecules administered) is given based on the mass of the
muscle rather than on the body weight of the individual and injected
within 3.0 cm of the area of the muscle containing the neuromuscular
junctions (based on ultrasound localization). Thus, for muscles like the
soleus where junctions are distributed along the length of muscle fibers,
toxin is delivered in multiple locations following the full length of the
muscle. In contrast, for muscles like the biceps brachii or medial and
lateral head of the gastrocnemius the injection pattern is an inverted U
shape following the distribution of the neuromuscular junctions. For
example, where 75U is sufficient to produce blockade of the neuromuscular
junctions in the lateral gastrocnemius, the biceps brachii has a mass 22%
larger than the lateral gastrocnemius, therefore requiring 91U for
efficacy. These absolute amounts are then diluted relative to increasing
mass and injected adjacent the neuromuscular junctions in the target
muscle.

[0040] Table 1 provides a multiplication factor by which the appropriate
dosage for other muscles may be determined. For example, the soleus
muscle has a mass approximately 2.63 times greater than the lateral
gastrocnemius. Where 75U of toxin is efficacious for relaxation of the
lateral gastrocnemius, and approximately 0.8 ml of a 100 U/ml
concentration of toxin is administered in a patient with a body weight of
up to 5 kg, approximately 2.0 ml (i.e., 2.63 times 0.8 ml) is efficacious
for relaxation of the soleus. Note that as body weight doubles to 10 kg,
20 kg, and 40 kg, toxin is diluted accordingly but injected in sufficient
volume such that the absolute amount of botulinum toxin administered
remains the same regardless of muscle size. Increasing muscle mass does
not require additional toxin because the number of neuromuscular
junctions does not change.

[0041]FIG. 4 shows a unipennate muscle 10 with a single transverse band
of neuromuscular junctions 50. Intramuscular injection of toxin is most
efficacious when delivered within 3.0 cm of this band. Similarly FIG. 5
shows unipennate gracilis muscle 11 with two transverse bands of
neuromuscular junctions 50. FIG. 6 shows a bipennate converging biceps
brachii muscle 12 having neuromuscular junctions 50 located in an
inverted "U" shape. FIG. 7 shows a rectus femoris muscle 13 with two
bands of neuromuscular junctions 50 running along its length, and FIG. 8
shows a deltoid muscle 14 with an irregular pattern of neuromuscular
junctions 50.

[0042] The principles, preferred embodiments and modes of operation of the
present invention have been described in the foregoing specification.
However, the invention should not be construed as limited to the
particular embodiments which have been described above. Instead, the
embodiments described here should be regarded as illustrative rather than
restrictive. Variations and changes may be made by others without
departing from the scope of the present invention as defined by the
following claims: